Culturing Hela cells

Passaging

• Remove and discard culture medium using the vacusafe and pasteur pipette. Rinse the cells with PBS (around 10mL) and remove it. This is because the Trypsin is inactivated by FBS in the media.
• Add 2.0 mL of Trypsin-EDTA solution to flask and put it back in the incubator and wait about 5 minutes. Tap the flask and observe cells under an inverted microscope until cell layer is dispersed.
• Add 8.0 mL of complete growth medium, this will stop the action of trypsin. Pipette up and down (gently) a few times to break the clusters of cells.
• If you are keeping the same flask:
  • Pipette away and discard a certain amount of media. The exact amount depends on the desired density of the passaged cells.
  • Add fresh media to the flask.
• If you are moving to a new flask:
  • Pipette the amount of cells you want in a new flask (less than the total if you want to decrease the density of cells in your flask).
  • Add some media to the new flask (12 - 15 mL in T75 or 5 mL in T25 - total).
• Put your name (if not already present) the date and increase the passage number on the flask and put them back in the incubator.